A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.

Mutations in a number of chromatin modifiers are associated with human neurological disorders. KDM5C, a histone H3 lysine 4 di- and tri-methyl (H3K4me2/3)-specific demethylase, is frequently mutated in X-linked intellectual disability (XLID) patients. Here, we report that disruption of the mouse Kdm5c gene recapitulates adaptive and cognitive abnormalities observed in XLID, including impaired social behavior and memory, and aggression. Kdm5c-knockout brains exhibit impaired dendritic arborization, spine abnormalities, and altered transcriptomes. In neurons, Kdm5c is recruited to promoters that harbor CpG islands decorated with high levels of H3K4me3, where it fine-tunes H3K4me3 levels. Kdm5c predominantly represses these genes, which include members of key pathways that regulate the development and function of neuronal circuitries. In summary, our mouse behavioral data strongly suggests that KDM5C mutations are causal to XLID. Furthermore, our findings suggest that loss of KDM5C function may impact gene expression in multiple regulatory pathways relevant to the clinical phenotypes.

CAAX proteins play essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis 1. The ~120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases 2. Proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of their C-terminal CAAX motifs 3 (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve cysteine prenylation, -AAX tripeptide cleavage, and methylation of the carboxyl prenylated Cys residue. The major CAAX protease activity is mediated by the Ras and a-factor converting enzyme 1 (Rce1), an integral membrane protease of the endoplasmic reticulum 4,5. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here, we report the crystal structure of a Methanococcus maripaludis homolog of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site, are distinct from other intramembrane proteases (IMPs). Catalytic residues are located ~10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. The farnesyl lipid is proposed to bind to a site at the opening of two transmembrane α-helices, which then positions the scissile bond adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.

The anaphase‐promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C‐terminal block of TPR motifs interacts with Hcn1, whereas an N‐terminal TPR block mediates Cdc16/Cut9 self‐association through a homotypic interface. This dimer interface is structurally related to the N‐terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo‐dimers through a conserved mechanism. The acetylated N‐terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N‐acetyl‐Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin‐dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.

BackgroundTherapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Molecular studies have shown few high-frequency mutations to be associated with endocrine resistance. In contrast, expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting.MethodsTo identify common adaptive mechanisms associated with resistance to aromatase inhibitors (AIs), we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44 and ZR75.1) or in 3D on collagen (MCF7) to model the stromal compartment. Furthermore, dimethyl labelling followed by LC-MS/MS was used to assess global changes in protein abundance. The role of target genes/proteins on proliferation, ER-mediated transcription and recruitment of ER to target gene promoters was analysed.ResultsThe cholesterol biosynthesis pathway was the common upregulated pathway in the ER+ LTED but not the ER– LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a 30–50 % drop in proliferation. Further analysis showed increased expression of 25-hydroxycholesterol (HC) in the MCF7 LTED cells. Exogenous 25-HC or 27-HC increased ER-mediated transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER– LTED cells. Additionally, recruitment of the ER and CREB-binding protein (CBP) to the TFF1 and GREB1 promoters was increased upon treatment with 25-HC and 27-HC. In-silico analysis of two independent studies of primary ER+ BC patients treated with neoadjuvant AIs showed that increased expression of MSMO1, EBP, LBR and SQLE enzymes, required for cholesterol synthesis and increased in our in-vitro models, was significantly associated with poor response to endocrine therapy.ConclusionTaken together, these data provide support for the role of cholesterol biosynthesis enzymes and the cholesterol metabolites, 25-HC and 27-HC, in a novel mechanism of resistance to endocrine therapy in ER+ BC that has potential as a therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0713-5) contains supplementary material, which is available to authorized users.

SummaryTFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.

A protocol combining immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition has been developed for enhanced analysis of protein phosphorylation. Immobilized metal ion affinity chromatography was initially used to enrich for phosphorylated peptides. Beta-elimination, with or without concurrent Michael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound to the chromatography resin. Derivatization of the phosphate facilitated the precise determination of phosphorylation sites by MALDI-PSD/LIFT tandem mass spectrometry, avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition in this manner circumvented several inherent disadvantages of the individual methods. In particular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to beta-elimination/Michael addition and (ii) the elution of peptides from the chromatography resin as derivatized phosphopeptides distinguished them from unphosphorylated species that were also retained. The chemical derivatization of phosphopeptides greatly increased the information obtained during peptide sequencing by mass spectrometry. The combined protocol enabled the detection and sequencing of phosphopeptides from protein digests at low femtomole concentrations of initial sample and was employed to identify novel phosphorylation sites on the cell adhesion protein p120 catenin and the glycoprotein fetuin.

Microchip-based field asymmetric waveform ion mobility spectrometry (FAIMS) analyzers featuring a grid of 35 µm -wide channels have allowed electric field intensity (E) over 60 kV/cm, or about twice that in previous devices with >0.5 mm gaps. Since the separation speed scales as E 4 to E 6 , these chips filter ions in just ~20 µs (or ~100 -10,000 times faster than "macroscopic" designs), although with reduced resolution. Here we report integration of these chips into electrospray ionization (ESI) mass spectrometry, with ESI coupled to FAIMS via a curtain plate/orifice interface with edgewise ion injection into the gap. Adjusting gas flows in the system permits control of ion residence time in FAIMS, which affects resolving power independently of ion desolvation after the ESI source. The results agree with a priori simulations and scaling rules. Applications illustrated include analyses of amino acids and peptides. Because of limited resolving power, the present FAIMS units are suitable to distinguish compound classes more than individual species. In particular, peptides separate from many other classes, including PEGs that are commonly encountered in proteomic analyses. In practical analyses with realistic time constraints, the effective separation power of present FAIMS may approach that of "macroscopic" systems.